Transforming growth factor-alpha (TGF-alpha) is a secretory mitogenic polypeptide predominantly expressed in various tumor-derived, retrovirally transformed and chemically-transformed cells, but expressed also in certain normal cells. TGF-alpha is structurally and functionally related to epidermal growth factor (EGF). These two factors are similar in their ability to bind to and activate the same receptor in mammalian cells but unlike TGF-alpha, EGF expression does not appear to be selectively induced in oncogenically transformed cells. An important feature of TGF-alpha and related factors is that they are synthesized as part of transmembrane glycoprotein precursors. Our general goal is to understand the biological properties of the membrane-bound precursor for TGF-alpha (pro TGF-alpha), how production and release of TGF-alpha from this precursor are regulated, and how these could be manipulated to control the release of TGF-alpha. Our work has demonstrated the membrane glycoprotein nature of proTGF-alpha. Transformed cells expressing this product have a limited capacity to generate soluble TGF-alpha which leads to accumulation of proTGF-alpha on the cell surface. We have shown that cell surface proTGF-alpha can establish contact with its receptor (the EGF receptor) on adjacent cells, an interaction that can support cell-cell adhesion and may lead to mitogenic stimulation. Furthermore, our recent evidence shows that the conversion of proTGF-alpha to mature TGF-alpha is rapidly activated by serum factors, tumor promoter phorbol esters and calcium ionophores. Based on the new findings, the availability of specific reagents and assays in our laboratory, we plan to investigate several important issues raised by the recent progress. These issues include: 1) whether signaling via the EGF receptor (EGFR) mediates the mitogenic response that is observed when cells expressing EGFR bind to cells expressing proTGF-alpha; 2) whether membrane precursors for other members of this growth factor family, proEGF and proVGF, will mimic proTGF-alpha in its ability to establish cell-cell contact via EGFR; 3) whether receptor-bound proTGF-alpha is cleaved and internalized upon binding to EGFR. We will develop an assay for proteases that cleave specific proTGF-alpha sequences. 4) we will seek evidence for transmembrane signaling by proTGF-alpha. We will explore the possibility that engagement with cell-bound or soluble EGFR has an effect on cells that express proTGF-alpha, specifically on DNA replication, expression of fast response genes, and autoregulation of proTGF-alpha gene expression in these cells. Finally, 5) we will determine the mechanism for activation of proTGF-alpha processing by various protein kinase-dependent pathways. These studies should provide insight into the a mode of communication between adjacent cells via a membrane-bound growth factor and its receptor.